Interferons (IFNs) are a well-known family of cytokines secreted by a large variety of eukaryotic cells. Interferons have a variety of biological activities, including anti-viral, immunomodulatory, immunoregulatory, neoplastic, and anti-proliferative properties, and have been utilized as therapeutic agents for treatment of diseases such as cancer, and various viral diseases. Interferons have demonstrated utility in the treatment of a variety of diseases, and are in widespread use for the treatment of multiple sclerosis and viral hepatitis; the most common therapeutic applications are currently treatment of hepatitis C and multiple sclerosis. Interferons are members of the growth hormone (GH) supergene family (Bazan, F. Immunology Today 11: 350-354 (1990); Mott, H. R. and Campbell, I. D. Current Opinion in Structural Biology 5: 114-121 (1995); Silvennoinen, O. and Ihle, J. N. (1996) SIGNALING BY THE HEMATOPOIETIC CYTOKINE RECEPTORS) which represents a set of proteins with similar structural characteristics. Each member of this family of proteins comprises a four helical bundle. While there are still more members of the family yet to be identified, some members of the family include the following: growth hormone, prolactin, placental lactogen, erythropoietin (EPO), thrombopoietin (TPO), interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12 (p35 subunit), IL-13, IL-15, oncostatin M, ciliary neurotrophic factor, leukemia inhibitory factor, alpha interferon, beta interferon, gamma interferon, omega interferon, tau interferon, epsilon interferon, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF) and cardiotrophin-1 (CT-1) (“the GH supergene family”). Members of the GH supergene family have similar secondary and tertiary structures, despite the fact that they generally have limited amino acid or DNA sequence identity. The shared structural features allow new members of the gene family to be readily identified. Four helical bundle and interferon polypeptides are described in WO 2005/074650 entitled “Modified Human Four Helical Bundle Polypeptides and Their Uses,” WO 2005/074524 entitled “Modified Human Interferon Polypeptides and Their Uses,” WO 2006/133089 and WO 2006/133088 entitled “Improved Human Interferon Polypeptides and Their Uses,” which are all incorporated by reference herein in their entirety.
Interferons include a number of related proteins, such as interferon-alpha (IFN-α), interferon-beta (IFN-β), interferon-gamma IFN-γ) interferon-kappa (IFN-κ, also known as interferon-epsilon or IFN-ε), interferon-tau (IFN-τ), and interferon-omega (IFN-ω). These interferon proteins are produced in a variety of cell types: IFN-α (leukocytes), IFN-β (fibroblasts), IFN-γ (lymphocytes), IFN-ε or κ (keratinocytes), IFN-ω (leukocytes) and IFN-τ (trophoblasts). IFN-α, IFN-β, IFN-ε or κ, IFN-ω, and IFN-τ are classified as type I interferons, while IFN-γ is classified as a type II interferon. Interferon alpha is encoded by a multi-gene family, while the other interferons appear to each be coded by a single gene in the human genome. Furthermore, there is some allelic variation in interferon sequences among different members of the human population.
Interferons are relatively small, single-chain glycoproteins released by cells invaded by viruses or exposed to certain other substances. Interferons are presently grouped into three major classes, designated: 1) leukocyte interferon (interferon-alpha, α-interferon, IFN-α), 2) fibroblast interferon (interferon-beta, β-interferon, IFN-β), and 3) immune interferon (interferon-gamma, γ-interferon, IFN-γ). In response to viral infection, lymphocytes synthesize primarily α-interferon (with omega interferon, IFN-ω), while infection of fibroblasts usually induces production of β-interferon. IFNα and IFNβ share about 20-30 percent amino acid sequence homology. The gene for human IFN-β lacks introns, and encodes a protein possessing 29% amino acid sequence identity with human IFN-α, suggesting that IFN-α and IFN-β genes have evolved from a common ancestor (Taniguchi et al., Nature 285 547-549 (1980)). By contrast, IFN-γ is synthesized by lymphocytes in response to mitogens. Pestka et al. in Annu. Rev. Immunol. (2004) 22:929-79, which is incorporated by reference herein in its entirety, describes class 2 α-helical cytokines including interferons (IFN-α, -β, -ε, -κ, -ω, -δ, -τ, and -γ) as well as interferon-like molecules such as limitin, IL-28A, IL-28B, and IL-29 as well as the ligands, receptors, and signal transduction pathways employed by these molecules. The interferons have different species and many allelic variants. In additional, interferons with novel activities and mutant sequences have been isolated from cells from patients with various diseases.
Interferons were originally derived from naturally occurring sources, such as buffy coat leukocytes and fibroblast cells, optionally using inducing agents to increase interferon production. Interferons have also been produced by recombinant DNA technology. The cloning and expression of mature IFNβ is described by Goeddel et al., Nucleic Acids Res. 8, 4057 (1980).
Type-I interferons all appear to bind a common receptor, type I IFN-R, composed of IFNAR1 and IFNAR2 subunits. The exact binding mode and downstream signal transduction cascades differ somewhat among the type I interferons. However, in general, the JAK/STAT signal transduction pathway is activated following binding of interferon to the interferon receptor. STAT transcription factors then translocate to the nucleus, leading to the expression of a number of proteins with antiviral, antineoplastic, and immunomodulatory activities.
The properties of naturally occurring type I interferon proteins are not optimal for therapeutic use. Type I interferons induce injection site reactions and a number of other side effects. They are highly immunogenic, eliciting neutralizing and non-neutralizing antibodies in a significant fraction of patients. Interferons are poorly absorbed from the subcutaneous injection site and have short serum half-lives. Finally, type I interferons do not express solubly in prokaryotic hosts, thus necessitating more costly and difficult refolding or mammalian expression protocols.
Specific examples of commercially available IFN products include IFNγ-1b (ACTIMMUNE®), IFNβ-1a (AVONEX®, and REBIF®), IFNβ-1b (BETASERON®), IFN alfacon-1 (INFERGEN®), IFNα-2 (INTRONA®), IFNα-2a (ROFERON-A®), Peginterferon alfa-2a (PEGASYS®), and Peginterferon alfa-2b (PEG-INTRON®). Some of the problems associated with the production of PEGylated versions of IFN proteins are described in Wang et al. (2002) Adv. Drug Deliv. Rev. 54:547-570; and Pedder, S. C. Semin Liver Dis. 2003; 23 Suppl 1:19-22. Wang et al. characterized positional isomers of PEG-INTRON®, and Pedder et al. compared PEGASYS® with PEG-INTRON® describing the lability of the PEGylation chemistries used and effects upon formulation. PEGASYS® is comprised of nine identifiable isoforms, which specific isoforms differing in anti-viral activity (Foser et al., Pharmacogenomics J 2003; 3:312). Despite the number of IFN products currently available on the market, there is still an unmet need for interferon therapeutics. The present invention is directed to identification of interferon proteins with improved properties. A number of groups have generated modified interferons with improved properties; the references below are all expressly incorporated by reference in their entirety.
Cysteine-depleted variants have been generated to minimize formation of unwanted inter- or intra-molecular disulfide bonds (U.S. Pat. Nos. 4,518,584; 4,588,585; and 4,959,314, which are incorporated by reference in their entirety). Methionine-depleted variants have been generated to minimize susceptibility to oxidation (EPO 260350, which is incorporated by reference herein).
Interferons with modified activity have been generated (U.S. Pat. Nos. 6,514,729; 4,738,844; 4,738,845; 4,753,795; 4,766,106; WO 00/78266, which are incorporated by reference herein). U.S. Pat. Nos. 5,545,723 and 6,127,332, which are incorporated by reference herein, disclose substitution mutants of interferon beta at position 101. Chimeric interferons comprising sequences from one or more interferons have been made (Chang et. al. Nature Biotech. 17: 793-797 (1999), U.S. Pat. Nos. 4,758,428; 4,885,166; 5,382,657; 5,738,846, which are incorporated by reference). Substitution mutations to interferon beta at positions 49 and 51 have also been described (U.S. Pat. No. 6,531,122, which is incorporated by reference). Expression and generation of IFN beta variants and conjugates have been discussed in U.S. Pat. Nos. 7,144,574 and 6,531,111, which are incorporated by reference herein in their entirety. Modifications discussed included glycosylation sites that were introduced into IFN beta or removed from the polypeptide, substitutions near glycosylation sites, conjugation to lysine or cysteine residues, and introduction or removal of amino acids.
Interferon beta variants with enhanced stability have been discussed, in which the hydrophobic core was optimized using rational design methods (WO 00/68387, which is incorporated by reference). Alternate formulations that promote interferon stability or solubility have also been disclosed (U.S. Pat. Nos. 4,675,483; 5,730,969; 5,766,582; WO 02/38170, which are incorporated by reference).
Interferon beta muteins with enhanced solubility have been discussed, in which several leucine and phenylalanine residues are replaced with serine, threonine, or tyrosine residues (WO 98/48018, which is incorporated by reference). Other modifications to improve solubility are discussed in US 2005/0054053 which is incorporated by reference herein in its entirety.
Interferon alpha and interferon beta variants with reduced immunogenicity have been discussed (See WO 02/085941 and WO 02/074783, which are incorporated by reference).
Immunogenicity is a major limitation of current interferon (including but not limited to, interferon beta) therapeutics. Although immune responses are typically most severe for non-human proteins, even therapeutics based on human proteins, such as interferon beta, are often observed to be immunogenic. Immunogenicity is a complex series of responses to a substance that is perceived as foreign and may include production of neutralizing and non-neutralizing antibodies, formation of immune complexes, complement activation, mast cell activation, inflammation, and anaphylaxis. A number of patients develop neutralizing antibodies to IFN beta (Int. Arch. Allergy Immunol. 118:368 371, 1999). It has been shown that development of IFN beta-neutralizing antibodies decreases the biological response to IFN beta, and causes a trend towards decreased treatment effect (Neurol. 50:1266 1272, 1998). Neutralizing antibodies will likely also impede the therapeutic utility of IFN beta in connection with treatment of other diseases (Immunol. Immuther. 39:263 268, 1994).
Several factors can contribute to protein immunogenicity, including but not limited to the protein sequence, the route and frequency of administration, and the patient population. Aggregation has been linked to the immunogenicity of a related protein therapeutic, interferon alpha [Braun et. al. Pharm. Res. 1997 14: 1472-1478]. Another study suggests that the presence of DR15 MHC alleles increases susceptibility to neutralizing antibody formation; interestingly, the same alleles also confer susceptibility to multiple sclerosis [Stickler et. al. Genes Immun. 2004 5: 1-7].
As aggregation may contribute to the immunogenicity of interferons (particularly interferon beta), variants engineered for improved solubility may also possess reduced immunogenicity. Cysteine-depleted variants have been generated to minimize formation of unwanted inter- or intra-molecular disulfide bonds (U.S. Pat. Nos. 4,518,584; 4,588,585; 4,959,314, which are incorporated by reference); such variants show a reduced propensity for aggregation. Interferon beta variants with enhanced stability have been made, in which the hydrophobic core was optimized using rational design methods (WO 00/68387, which is incorporated by reference); in some cases solubility may be enhanced by improvements in stability. Alternate formulations that promote interferon stability and solubility have also been disclosed (U.S. Pat. Nos. 4,675,483; 5,730,969; 5,766,582; WO 02/38170, which are incorporated by reference). Interferon beta muteins with enhanced solubility have been discussed, in which several leucine and phenylalanine residues are replaced with serine, threonine, or tyrosine residues (WO 98/48018, which is incorporated by reference).
Interferons have been modified by the addition of polyethylene glycol (“PEG”) (see U.S. Pat. Nos. 4,917,888; 5,382,657; and 6,962,978; WO 99/55377; WO 02/09766; WO 00/23114, all of which are incorporated by reference in their entirety). PEG addition can improve serum half-life and solubility. In some cases, PEGylation has been observed to reduce the fraction of patients who raise neutralizing antibodies by sterically blocking access to antibody agretopes (see for example, Hershfield et. al. PNAS 1991 88:7185-7189 (1991); Bailon. et al. Bioconjug. Chem. 12: 195-202 (2001); He et al. Life Sci. 65: 355-368 (1999)).
Interferon beta variants have also been generated that are predicted to bind class II MHC alleles with decreased affinity relative to the wild type protein; in both examples primarily alanine mutations were used to confer decreased binding [WO 02/074783, which is incorporated by reference; Stickler supra]. Immunoreactivity of antibodies against synthetic peptides corresponding to portions of IFN beta have been discussed in Redlich et al. Proc. Natl. Acad. Sci. (1991) 88:4040-4044.
Several methods have been developed to modulate the immunogenicity of proteins; a preferred approach is to disrupt T-cell activation by removing MHC-binding agretopes. This approach is more tractable than evading T-cell receptor or antibody binding, as the diversity of MHC molecules comprises only ˜103 alleles, while the antibody repertoire is estimated to be approximately 108 and the T-cell receptor repertoire is larger still. By identifying and removing or modifying class II MHC-binding peptides within a protein sequence, the molecular basis of immunogenicity can be evaded. The elimination of such agretopes for the purpose of generating less immunogenic proteins has been disclosed previously; see for example WO 98/52976 and WO 02/079232, which are incorporated by reference.
While a large number of mutations in MHC-binding agretopes may be identified that are predicted to confer reduced immunogenicity, most of these amino acid substitutions will be energetically unfavorable. As a result, the vast majority of the reduced immunogenicity sequences identified using the methods described above will be incompatible with the structure and/or function of the protein. In order for MHC agretope removal to be a viable approach for reducing immunogenicity, it is crucial that simultaneous efforts are made to maintain a protein's structure, stability, and biological activity.
Immunogenicity may limit the efficacy and safety of interferon therapeutics in multiple ways. Therapeutic efficacy may be reduced directly by the formation of neutralizing antibodies. Efficacy may also be reduced indirectly, as binding to either neutralizing or non-neutralizing antibodies may alter serum half-life. Unwanted immune responses may take the form of injection site reactions, including but not limited to delayed-type hypersensitivity reactions. It is also possible that anti-interferon beta neutralizing antibodies may cross-react with endogenous interferon beta and block its function.
There remains a need for novel interferon proteins having reduced immunogenicity. Variants of interferon with reduced immunogenicity could find use in the treatment of a number of interferon responsive conditions. U.S. Patent Publication No. 2005/0054053, which is incorporated by reference herein, describes variant IFN beta proteins with modulated immunogenicity as compared with wild-type IFN beta.
As a result, there exists a need for the development and discovery of interferon proteins with improved properties, including but not limited to increased efficacy, decreased side effects, decreased immunogenicity, increased solubility, and enhanced soluble prokaryotic expression. There is a need for interferon polypeptides that require less frequent injection and/or result in decreased risk of developing neutralizing antibodies. Improved interferon therapeutics could may be useful for the treatment of a variety of diseases and conditions, including autoimmune diseases, viral infections, and, inflammatory diseases, cell proliferation diseases, bacterial infections, enhancing fertility, and cancer, among others and transplant rejection. In addition, interferons may be used to promote the establishment of pregnancy in certain mammals.
The use of human interferon beta, one member of the interferon family, is best established in the treatment of multiple sclerosis. Two forms of recombinant interferon beta, have recently been licensed in Europe and the U.S. for treatment of this disease. One form is interferon-beta-1a (trademarked and sold as AVONEX®, mfg. Biogen, Inc., Cambridge, Mass.; or as REBIF®, mfg. Merck Serono) and hereinafter, “interferon-beta-1a” or “IFN-beta-1a” or “IFN-β-1a” or “interferon-β-1a”, or in various hyphenated and unhyphenated forms, used interchangeably. A currently marketed formulation of AVONEX® has 30 ug/dose (200 MIU/mg) and provides CHO-derived (Chinese Hamster Ovary) IFN-beta 1a given intramuscularly four times per week. A currently marketed formulation of REBIF® has 44 μg/dose (270 MIU/mg) given subcutanelous TIW and also provides CHO-derived IFN beta 1a. The other form is interferon-beta-1b (trademarked and sold as BETASERON® Berlex, Richmond, Calif.), hereinafter, “interferon-beta-1b”. A currently marketed formulation of BETASERON® has 250 μg/dose (32 MIU/mg) and provides E. Coli-derived IFN-beta 1b given subcutaneously every other day or three times daily. Interferon beta-1a is produced in mammalian cells using the natural human gene sequence and is glycosylated, whereas interferon beta-1b is produced in E. coli bacteria using a modified human gene sequence that contains a genetically engineered cysteine-to-serine substitution at amino acid position 17 (C17S) and is non-glycosylated. Common side effects include, but are not limited to, fever, headaches, fatigue, anxiety, depression, liver disorders, and injection site reactions. Yong et al. Neurology (1998) 51:682-689 discuss the use of interferon beta in the treatment of multiple sclerosis and indicate the accumulation rate of disability from MS is reduced.
The crystal structure of glycosylated human interferon beta has been described by Karpusas et al. in Proc Natl. Acad Sci 1997 94:11813-11818. This protein is glycosylated at a single site (Asn80). The protein has a tendency to aggregate when produced in E. coli (Mitsui et al. Pharmacol Ther 1993 58:93-132). Karpusas et al. describe the production of human interferon beta in Chinese hamster ovary cells (CHO) and purification of the secreted protein using blue Sepharose and SP-Sepharose (ion exchange).
Alpha and beta interferons have been used in the treatment of the acute viral disease herpes zoster (T. C. Merigan et al, N. Engl. J. Med. 298, 981-987 (1978); E. Heidemann et al., Onkologie 7, 210-212 (1984)), chronic viral infections, e.g. hepatitis C and hepatitis B infections (R. L. Knobler et al., Neurology 34(10): 1273-9 (1984); M. A. Faerkkilae et al., Act. Neurol. Sci. 69, 184-185 (1985)).
Human IFNβ is a regulatory polypeptide with a molecular weight of about 22 kDa consisting of 166 amino acid residues. It can be produced by most cells in the body, in particular fibroblasts, in response to viral infection or exposure to other agents. It binds to a multimeric cell surface receptor, and productive receptor binding results in a cascade of intracellular events leading to the expression of IFNβ inducible genes which in turn produces effects which can be classified as anti-viral, anti-proliferative and immunomodulatory.
The amino acid sequence of human IFNβ is known (Taniguchi, Gene 10:11-15, 1980, and in EP 83069, EP 41313 and U.S. Pat. No. 4,686,191 which are incorporated by reference herein). Human and murine IFNβ crystal structures have been described in Proc. Natl. Acad. Sci. USA 94:11813-11818, 1997; J. Mol. Biol. 253:187-207, 1995; U.S. Pat. No. 5,602,232; 5,460,956; 5,441,734; 4,672,108; which are incorporated by reference herein and discussed in Cell Mol. Life. Sci. 54:1203-1206, 1998. Variants of IFMβ have been reported (WO 95/25170, WO 98/48018, U.S. Pat. No. 6,572,853, U.S. Pat. No. 5,545,723, U.S. Pat. No. 4,914,033, EP 260350, U.S. Pat. No. 4,588,585, U.S. Pat. No. 4,769,233, Stewart et al, DNA Vol. 6 no. 2 1987 pp. 119-128, Runkel et al, 1998, J. Biol. Chem. 273, No. 14, pp. 8003-8008, which are incorporated by reference herein). U.S. Pat. No. 4,966,843, U.S. Pat. No. 5,376,567, U.S. Pat. No. 5,795,779, U.S. Pat. No. 7,144,574, which are incorporated by reference herein, describe the expression of IFNβ in CHO cells. IFNβ molecules with a particular glycosylation pattern and methods for their preparation have been reported (EP 287075 and EP 529300).
The structure and function of IFNβ1a and β1b have been compared in Pharmaceut. Res. 15:641-649, 1998. The progression of multiple sclerosis has been shown to be delayed with IFN beta. Multiple sclerosis is a relapsing then progressive inflammatory degenerative disease of the central nervous system. Other effects that IFNβ may have include, but are not limited to, inhibitory effects on the proliferation of leukocytes and antigen presentation, modulation of the profile of cytokine production towards an anti-inflammatory phenotype, and reduction of T-cell migration by inhibiting the activity of T-cell matrix metalloproteases to account for the mechanism of IFNβ in MS (Neurol. 51:682-689, 1998).
IFN beta may be used in the treatment of a number of diseases including, but not limited to, osteosarcoma, basal cell carcinoma, cervical dysplasia, glioma, acute myeloid leukemia, multiple myeloma, Hodgkin's disease, breast carcinoma, melanoma, and viral infections, including but not limited to, papilloma virus, viral hepatitis, herpes genitalis, herpes zoster, herpetic keratitis, herpes simplex, viral encephalitis, cytomegalovirus pneumonia, and rhinovirus. Side effects of current IFNβ therapeutics include injection site reactions, fever, chills, myalgias, arthralgias, and other flu-like symptoms (Clin. Therapeutics, 19:883-893, 1997).
An improved IFNβ-like molecule is needed, considering the multitude of side effects with current IFNβ products, their association with frequent injection, the risk of developing neutralizing antibodies impeding the desired therapeutic effect of IFNβ, and the potential for obtaining more optimal therapeutic IFNβ levels with concomitant enhanced therapeutic effect.
The relative in vitro potencies of interferon-beta-1a and interferon beta 1b in functional assays have been compared, and it was shown that the specific activity of interferon-beta-1a is approximately 10-fold greater than the specific activity of interferon-beta-1b (Runkel et al., 1998, Pharm. Res. 15: 641-649). From studies designed to identify the structural basis for these activity differences, glycosylation was identified as the only one of the known structural differences between the products that affected the specific activity. The effect of the carbohydrate was largely manifested through its stabilizing role on structure. The stabilizing effect of the carbohydrate was evident in thermal denaturation experiments and SEC analysis. Lack of glycosylation was also correlated with an increase in aggregation and an increased sensitivity to thermal denaturation. Enzymatic removal of the carbohydrate from interferon-beta-1a with PNGase F caused extensive precipitation of the deglycosylated product.
Interferon-beta molecules of the invention may retain all or most of their biological activities and the following properties may result: altered pharmacokinetics and pharmacodynamics leading to increased half-life and alterations in tissue distribution (e.g., ability to stay in the vasculature for longer periods of time), increased stability in solution, reduced immunogenicity, protection from proteolytic digestion and subsequent abolition of activity. Such molecules would be a substantial advance in the pharmaceutical and medical arts and would make a significant contribution to the management of various diseases in which interferon has some utility, such as multiple sclerosis, fibrosis, and other inflammatory or autoimmune diseases, cancers, hepatitis and other viral diseases. In particular, the ability to remain for longer periods of time in the vasculature allows the interferon beta to be used to inhibit angiogenesis and potentially to cross the blood-brain barrier. Conjugates formed between interferon beta comprising a non-naturally encoded amino acid and another molecule, including but not limited to a polymer, could result in modulated thermal stability of the conjugate. Such modulated thermal stability may be an advantage when formulating interferon-beta in powder form for use in subsequent administration via inhalation.
Covalent attachment of the hydrophilic polymer poly(ethylene glycol), abbreviated PEG, is a method of increasing water solubility, bio availability, increasing serum half-life, increasing therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending the circulation time of many biologically active molecules, including proteins, peptides, and particularly hydrophobic molecules. PEG has been used extensively in pharmaceuticals, on artificial implants, and in other applications where bio compatibility, lack of toxicity, and lack of immunogenicity are of importance. In order to maximize the desired properties of PEG, the total molecular weight and hydration state of the PEG polymer or polymers attached to the biologically active molecule must be sufficiently high to impart the advantageous characteristics typically associated with PEG polymer attachment, such as increased water solubility and circulating half life, while not adversely impacting the bioactivity of the parent molecule.
PEG derivatives are frequently linked to biologically active molecules through reactive chemical functionalities, such as lysine, cysteine and histidine residues, the N-terminus and carbohydrate moieties. Proteins and other molecules often have a limited number of reactive sites available for polymer attachment. Often, the sites most suitable for modification via polymer attachment play a significant role in receptor binding, and are necessary for retention of the biological activity of the molecule. As a result, indiscriminate attachment of polymer chains to such reactive sites on a biologically active molecule often leads to a significant reduction or even total loss of biological activity of the polymer-modified molecule. R. Clark et al., (1996), J. Biol. Chem., 271:21969-21977. To form conjugates having sufficient polymer molecular weight for imparting the desired advantages to a target molecule, prior art approaches have typically involved random attachment of numerous polymer arms to the molecule, thereby increasing the risk of a reduction or even total loss in bioactivity of the parent molecule.
Reactive sites that form the loci for attachment of PEG derivatives to proteins are dictated by the protein's structure. Proteins, including enzymes, are composed of various sequences of alpha-amino acids, which have the general structure H2N—CHR—COOH. The alpha amino moiety (H2N—) of one amino acid joins to the carboxyl moiety (—COOH) of an adjacent amino acid to form amide linkages, which can be represented as —(NH—CHR—CO)n—, where the subscript “n” can equal hundreds or thousands. The fragment represented by R can contain reactive sites for protein biological activity and for attachment of PEG derivatives.
For example, in the case of the amino acid lysine, there exists an —NH2 moiety in the epsilon position as well as in the alpha position. The epsilon —NH2 is free for reaction under conditions of basic pH. Much of the art in the field of protein derivatization with PEG has been directed to developing PEG derivatives for attachment to the epsilon —NH2 moiety of lysine residues present in proteins. “Polyethylene Glycol and Derivatives for Advanced PEGylation”, Nektar Molecular Engineering Catalog, 2003, pp. 1-17. These PEG derivatives all have the common limitation, however, that they cannot be installed selectively among the often numerous lysine residues present on the surfaces of proteins. This can be a significant limitation in instances where a lysine residue is important to protein activity, existing in an enzyme active site for example, or in cases where a lysine residue plays a role in mediating the interaction of the protein with other biological molecules, as in the case of receptor binding sites.
A second and equally important complication of existing methods for protein PEGylation is that the PEG derivatives can undergo undesired side reactions with residues other than those desired. Histidine contains a reactive imino moiety, represented structurally as —N(H)—, but many chemically reactive species that react with epsilon —NH2 can also react with —N(H)—. Similarly, the side chain of the amino acid cysteine bears a free sulfhydryl group, represented structurally as —SH. In some instances, the PEG derivatives directed at the epsilon —NH2 group of lysine also react with cysteine, histidine or other residues. This can create complex, heterogeneous mixtures of PEG-derivatized bioactive molecules and risks destroying the activity of the bioactive molecule being targeted. It would be desirable to develop PEG derivatives that permit a chemical functional group to be introduced at a single site within the protein that would then enable the selective coupling of one or more PEG polymers to the bioactive molecule at specific sites on the protein surface that are both well-defined and predictable.
In addition to lysine residues, considerable effort in the art has been directed toward the development of activated PEG reagents that target other amino acid side chains, including cysteine, histidine and the N-terminus. See, e.g., U.S. Pat. No. 6,610,281 which is incorporated by reference herein, and “Polyethylene Glycol and Derivatives for Advanced PEGylation”, Nektar Molecular Engineering Catalog, 2003, pp. 1-17. A cysteine residue can be introduced site-selectively into the structure of proteins using site-directed mutagenesis and other techniques known in the art, and the resulting free sulfhydryl moiety can be reacted with PEG derivatives that bear thiol-reactive functional groups. This approach is complicated, however, in that the introduction of a free sulfhydryl group can complicate the expression, folding and stability of the resulting protein. Thus, it would be desirable to have a means to introduce a chemical functional group into bioactive molecules that enables the selective coupling of one or more PEG polymers to the protein while simultaneously being compatible with (i.e., not engaging in undesired side reactions with) sulfhydryls and other chemical functional groups typically found in proteins.
As can be seen from a sampling of the art, many of these derivatives that have been developed for attachment to the side chains of proteins, in particular, the —NH2 moiety on the lysine amino acid side chain and the —SH moiety on the cysteine side chain, have proven problematic in their synthesis and use. Some form unstable linkages with the protein that are subject to hydrolysis and therefore decompose, degrade, or are otherwise unstable in aqueous environments, such as in the bloodstream. Some form more stable linkages, but are subject to hydrolysis before the linkage is formed, which means that the reactive group on the PEG derivative may be inactivated before the protein can be attached. Some are somewhat toxic and are therefore less suitable for use in vivo. Some are too slow to react to be practically useful. Some result in a loss of protein activity by attaching to sites responsible for the protein's activity. Some are not specific in the sites to which they will attach, which can also result in a loss of desirable activity and in a lack of reproducibility of results. In order to overcome the challenges associated with modifying proteins with poly(ethylene glycol) moieties, PEG derivatives have been developed that are more stable (e.g., U.S. Pat. No. 6,602,498, which is incorporated by reference herein) or that react selectively with thiol moieties on molecules and surfaces (e.g., U.S. Pat. No. 6,610,281, which is incorporated by reference herein). There is clearly a need in the art for PEG derivatives that are chemically inert in physiological environments until called upon to react selectively to form stable chemical bonds.
Recently, an entirely new technology in the protein sciences has been reported, which promises to overcome many of the limitations associated with site-specific modifications of proteins. Specifically, new components have been added to the protein biosynthetic machinery of the prokaryote Escherichia coli (E. coli) (e.g., L. Wang, et al., (2001), Science 292:498-500) and the eukaryote Sacchromyces cerevisiae (S. cerevisiae) (e.g., J. Chin et al., Science 301:964-7 (2003)), which has enabled the incorporation of non-genetically encoded amino acids to proteins in vivo. A number of new amino acids with novel chemical, physical or biological properties, including photoaffinity labels and photoisomerizable amino acids, photocrosslinking amino acids (see, e.g., Chin, J. W., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99:11020-11024; and, Chin, J. W., et al., (2002) J. Am. Chem. Soc. 124:9026-9027), keto amino acids, heavy atom containing amino acids, and glycosylated amino acids have been incorporated efficiently and with high fidelity into proteins in E. coli and in yeast in response to the amber codon, TAG, using this methodology. See, e.g., J. W. Chin et al., (2002), Journal of the American Chemical Society 124:9026-9027; J. W. Chin, & P. G. Schultz, (2002), Chem Bio Chem 3(11):1135-1137; J. W. Chin, et al., (2002), PNAS United States of America 99:11020-11024; and, L. Wang, & P. G. Schultz, (2002), Chem. Comm., 1:1-11. All references are incorporated by reference in their entirety. These studies have demonstrated that it is possible to selectively and routinely introduce chemical functional groups, such as ketone groups, alkyne groups and azide moieties, that are not found in proteins, that are chemically inert to all of the functional groups found in the 20 common, genetically-encoded amino acids and that may be used to react efficiently and selectively to form stable covalent linkages.
The ability to incorporate non-genetically encoded amino acids into proteins permits the introduction of chemical functional groups that could provide valuable alternatives to the naturally-occurring functional groups, such as the epsilon —NH2 of lysine, the sulfhydryl —SH of cysteine, the imino group of histidine, etc. Certain chemical functional groups are known to be inert to the functional groups found in the 20 common, genetically-encoded amino acids but react cleanly and efficiently to form stable linkages. Azide and acetylene groups, for example, are known in the art to undergo a Huisgen [3+2] cycloaddition reaction in aqueous conditions in the presence of a catalytic amount of copper. See, e.g., Tornoe, et al., (2002) J. Org. Chem. 67:3057-3064; and, Rostovtsev, et al., (2002) Angew. Chem. Int. Ed. 41:2596-2599. By introducing an azide moiety into a protein structure, for example, one is able to incorporate a functional group that is chemically inert to amines, sulfhydryls, carboxylic acids, hydroxyl groups found in proteins, but that also reacts smoothly and efficiently with an acetylene moiety to form a cycloaddition product. Importantly, in the absence of the acetylene moiety, the azide remains chemically inert and unreactive in the presence of other protein side chains and under physiological conditions.
The present invention addresses, among other things, problems associated with the activity and production of IFN beta polypeptides, and also addresses the production of a IFN beta polypeptide with improved biological or pharmacological properties, such as enhanced antiviral activity and/or improved therapeutic half-life.